Glucose sensor molecules

ABSTRACT

The present invention provides a glucose sensor having a glucose receptor containing a binding site of formula (I): wherein X, n, m and R 1  are defined herein. Also provided is a glucose sensor molecule for use in such a glucose sensor, the glucose sensor molecule containing the binding site of formula (I). The binding site has been found to have particularly good selectivity for glucose.

The invention relates to glucose sensors containing a glucose receptorhaving a particular glucose binding site. Glucose sensor moleculescontaining the glucose binding site are also provided.

BACKGROUND TO THE INVENTION

The monitoring of glucose levels is of vital importance in the clinicalsetting. In particular, the regular monitoring of tissue glucoseconcentrations by diabetic patients and the care of hypoglycemicpatients in an intensive care environment require simple and reliablemethods for monitoring glucose levels. Such glucose monitoring hasusually been based on electrochemical technology and glucose selectiveenzymes such as glucose oxidase. Sensors based on this technology aresusceptible to denaturing of the enzyme, particularly in a biologicalenvironment. Further, because they are consumptive of glucose and relyon constant diffusion of glucose to the sensor electrodes, they aresusceptible to errors and drift.

An alternative technology to the electrochemical devices is the use ofoptical sensors, such as those based on fluorescence intensitymeasurements. For instance, reversible, non-consumptive fluorescentoptical sensors utilizing fluorophore boronic acid chemistries as theindicator for glucose have been developed. Such sensors measure thechange in the emitted fluorescent intensity as a means of determiningglucose concentration. Such boronic acid glucose indicating chemistrieshave the advantage of being reversible with glucose, non-consumptive andare more stable than the enzymes, such as glucose oxidase, which arecommonly used in electrochemical glucose sensors. They can also bereadily immobilized, within a hydrogel, onto an optical fibre.

These sensors rely on the selective binding of glucose to the boronicacid binding site. Boronic acids, however, are capable of binding othersaccharides, for example galactose and fructose. An effective sensorshould therefore provide good selectivity for glucose over othersaccharides.

SUMMARY OF THE INVENTION

The present invention provides a glucose sensor having a particularglucose binding site which has been found to have a high selectivity forglucose over other saccharides. Sensors selective for glucose and othersaccharides have been previously disclosed, for example in U.S. Pat. No.6,387,672. U.S. Pat. No. 6,387,672 describes sensors having a bindingsite of formula:

wherein the Bd₁ and Bd₂ groups are binding groups such as boronic acidsand Sp represents an aliphatic spacer. The length of the carbon chain inthe aliphatic spacer is selected to match the nature of the analyte. Forbinding to glucose, U.S. Pat. No. 6,387,672 teaches the use of astraight chain 6-carbon atom aliphatic spacer which is said to providegood selectivity for glucose.

The present inventors, however, have found that the selectivity of thebinding site for glucose can be further improved by altering the6-carbon atom aliphatic spacer. The present invention therefore providesa glucose sensor comprising a glucose receptor having a binding site offormula (I):

wherein X represents O, S, NR₂ or CHR₃;n is from 1 to 4;m is from 1 to 4, and n+m is 5;R₂ represents hydrogen or C₁₋₄ alkyl;each R₁ is the same or different and represents hydrogen, C₁₋₄ alkyl orC₃₋₇ cycloalkyl;or R₁, together with an adjacent R₁, R₂ or R₃ group and the carbon ornitrogen atoms to which they are attached, form a C₃₋₇ cycloalkyl or a5- or 6-membered heterocyclyl group,wherein when X represents CHR₃, R₃ together with an adjacent R₁ groupand the carbon atoms to which they are attached form a C₃₋₇ cycloalkylgroup.

Particularly preferred receptors are those wherein X represents O, S orNR₂, preferably O or NH, in particular O.

Also provided is a glucose sensor molecule of formula (II):

wherein X, n, m and R₁ are as defined above;Fl is a fluorophore;L₁ and L₂ are the same or different and represent a linker; andR₄ is a support material, a hydrogen atom or an anchor group suitablefor attaching the sensor molecule to a support material.

The present invention also provides a process for the preparation of aglucose sensor molecule as set out above, which process comprisesreductive amination of (III) in the presence of (IV), followed bydeprotection of the boronic acid group and optionally deprotection ofR₄:

Fl-L₁-NH—(CHR₁)_(n)—X—(CHR₁)_(m)—NH-L₂-R₄  (III)

wherein X, n, m, R₁ Fl, L₁ and L₂ are as defined above; andR₄ is a hydrogen atom or an anchor group suitable for attaching thesensor molecule to a support material, wherein R₄ is optionallyprotected by a protecting group;

wherein B(PG) is a boronic acid group protected by a protecting group.

Also provided is a method of detecting or quantifying the amount ofglucose in an analyte, the method comprising contacting the analyte witha glucose receptor comprising a binding site of formula (I):

wherein X, n, m and R₁ are as defined above.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a graph of the relative fluorescence intensity versuscarbohydrate concentration for glucose sensor compound 18 of theinvention in the presence of D-glucose, D-fructose, D-galactose andD-mannose.

FIGS. 2 and 3 provide similar graphs of the relative fluorescenceintensity versus carbohydrate concentration for two comparativecompounds.

DETAILED DESCRIPTION OF THE INVENTION

As used herein a C₁₋₄ alkyl group may be a straight chain or branchedalkyl group, for example a t-butyl, n-butyl, i-propyl, n-propyl, ethylor methyl group, e.g. ethyl or methyl. C₁₋₄ alkyl groups are typicallyunsubstituted.

As used herein a C₁₋₆ alkylene group may be a straight chain or branchedalkylene group, but is typically a straight chain alkylene group. A C₁₋₆alkylene group is typically a C₁₋₄ alkylene group, for examplen-butylene, n-propylene, ethylene or methylene, e.g. ethylene ormethylene. C₁₋₆ alkylene groups are typically unsubstituted.

As used herein a C₃₋₇ cycloalkyl group is typically a cyclopentyl orcyclohexyl group. C₃₋₇ cycloalkyl groups may be unsubstituted orsubstituted. Suitable substituents are C₁₋₄ alkyl groups, for examplemethyl and ethyl. Preferably, a C₃₋₇ cycloalkyl group is unsubstituted.

As used herein a 5- or 6-membered heterocyclyl group is a 5- or6-membered saturated ring containing one or more, typically one or two,e.g. one, heteroatom selected from N, O and S. Preferred heterocyclylgroups are those containing a nitrogen atom, for example piperidinyl andpyrrolidinyl. Heterocyclyl groups may be unsubstituted or substituted.Suitable substituents are C₁₋₄ alkyl groups, for example methyl andethyl. Preferably, a heterocyclyl group is unsubstituted.

As used herein an arylene group is an unsaturated group which may bemonocyclic, bicyclic, or which may contain three or four fused rings.Typically, an arylene group is phenylene. Arylene groups may beunsubstituted or substituted. Suitable substituents are C₁₋₄ alkylgroups, for example methyl and ethyl. Preferably, an arylene group isunsubstituted.

The present invention relates to glucose sensors, in particularfluorescent sensors. The sensor comprises a glucose receptor having abinding site having two boronic acid groups separated by a specificspacer group. Glucose present in the analyte binds to the boronic acidgroups and forms a 1:1 complex with the receptor.

In the case of a fluorescent sensor, the sensor also comprises afluorophore which is associated with the glucose receptor. Where afluorophore is associated with the receptor, this indicates that bindingof a glucose molecule to the receptor perturbs the fluorescence of thefluorophore, e.g. its wavelength, intensity or lifetime. Typically, inthe absence of glucose, the receptor acts to quench the fluorescence ofthe fluorophore. However, where glucose is bound to the fluorophore,fluorescence quenching no longer occurs so that the intensity of thefluorescence is increased. Such fluorescent sensors therefore exhibit achange in wavelength, intensity and/or lifetime of the fluorescence whenglucose binds to the binding site. Thus, the sensor may detect orquantify the amount of glucose present in the analyte by monitoringchanges in the wavelength, intensity and/or lifetime of thefluorescence. Typically, the intensity or the lifetime is measured.

The glucose binding site of the present invention comprises a spacerbetween the two nitrogen atoms which is chosen to provide improvedselective binding to glucose. The spacer has the formula:

—(CHR₁)_(n)—X—(CHR₁)_(m)—

X represents either a heteroatom selected from O, S or NR₂ or X mayrepresent CHR₃. Typically, X represents O, S or NR₂, preferably Xrepresents O.

The spacer is a 6-membered chain. Therefore, whilst n and m may varybetween 1 and 4, the total n+m is always 5. Preferably, n is 2 or 3 andm is 2 or 3.

Typically, each R₁ is the same or different and is selected fromhydrogen, C₁₋₄ alkyl and C₃₋₇ cycloalkyl, preferably from hydrogen andC₁₋₄ alkyl, more preferably from hydrogen, methyl and ethyl. Mostpreferably R₁ is hydrogen. The spacer group contains five R₁ groups.Typically, at least four of the R₁ groups represent hydrogen. In apreferred embodiment, all R₁ groups are the same and represent hydrogen.

In one embodiment, the spacer comprises a cyclic group. In thisembodiment, R₁, together with an adjacent R₁, R₂ or R₃ group and thecarbon or nitrogen atoms to which they are attached, form a C₃₋₇cycloalkyl or 5- or 6-membered heterocyclyl group. The binding site inthis embodiment is typically of formula (Ia):

wherein p is from 1 to 4;q is from 0 to 3, and p+q is 4;X is N or CH, preferably X is N;each R₁ is the same or different and represents hydrogen, C₁₋₄ alkyl orC₃₋₇ cycloalkyl; andring A is a C₃₋₇ cycloalkyl group or a 5- to 7-membered heterocyclylgroup.

In formula (Ia), typically, each R₁ is the same or different and isselected from hydrogen, C₁₋₄ alkyl and C₃₋₇ cycloalkyl, preferably fromhydrogen and C₁₋₄ alkyl, more preferably from hydrogen, methyl andethyl. Most preferably R₁ is hydrogen. Preferably, at least 3 of the R₁groups present represent hydrogen. More preferably, all R₁ groups arethe same and represent hydrogen.

In formula (Ia), preferably p is 2 or 3 and q is 1 or 2, and p+q is 4.

When X is N, ring A typically forms a 5- or 6-membered heterocyclylgroup, typically a piperidinyl or pyrrolidinyl group. When X is CH, ringA typically forms a C₃₋₇ cycloalkyl group, preferably cyclopentyl orcyclohexyl.

The cycloalkyl and heterocyclyl groups may be unsubstituted orsubstituted. Suitable substituents are C₁₋₄ alkyl groups. Preferably,the cycloalkyl and heterocyclyl groups are unsubstituted.

In a particularly preferred embodiment, in the binding site of formula(I):

X represents O;n is from Ito 4;m is from 1 to 4 and n+m is 5; andeach R₁ represents hydrogen.

In a more preferred embodiment, in the binding site of formula (I):

X represents O;n is 2 or 3, m is 2 or 3 and n+m is 5; andeach R₁ represents hydrogen.

Thus, particularly preferred binding sites of formula (I) are those offormula (I′) and (I″), with (I″) being most preferred:

The two nitrogen atoms in the receptor marked as N* below:

may either bear a hydrogen atom or may be connected to further moieties,e.g. to a fluorophore or to a support material. The moieties bonded tothe two nitrogen atoms may be the same or different, but are typicallydifferent.

The receptor may be bonded via one of the nitrogen atoms to a supportmaterial. A support material as used herein is a molecule or substanceto which the receptor can be tethered. The support material typicallyserves to immobilise or to restrict the movement of the receptor withinthe sensor. In one embodiment, the support material is a solid orgel-like support material such as a polymeric matrix. This can be usedto physically immobilise the receptor in the desired position within thesensor. A hydrogel (a highly hydrophilic cross-linked polymeric matrixsuch as a cross-linked polyacrylamide) is an example of a suitablepolymeric matrix.

In an alternative embodiment, the support material may be awater-soluble polymer or other water-soluble molecule such that thereceptor-support material complex is itself water-soluble. Such awater-soluble complex may be provided in aqueous solution within thesensor. Examples of suitable water-soluble polymers include linear orlightly cross-linked polyacrylamides or polyvinyl alcohols. Otherwater-soluble molecules which can be used as the support materialinclude dendrimers, cyclodextrins, cryptans and crown ethers. Dendrimersare preferred.

The sensor typically comprises a membrane to restrict or prevent theleakage of the receptor out of the sensor, but which allows glucose toenter the sensor. Dialysis membranes are suitable for this purpose. Theuse of a water-soluble support material serves to increase the molecularweight of the receptor and may also increase its water-solubility. Theincrease in molecular weight assists in restricting the passage of thereceptor through the membrane and thereby restricts the movement of thereceptor. The support material is typically of high molecular weight,for example at least 1000, preferably at least 2000, 5000 or 10,000.

Linkers may be used to connect the receptor to the support material.Examples of suitable linkers are alkylene or arylene groups, orcombinations thereof, as described further below.

The presence of a support material on one of the nitrogen atoms is notessential. For example, where the receptor is itself water-soluble andof sufficiently high molecular weight that it does not pass through adialysis membrane, no further attachment of support material is needed.In this case, the nitrogen atom may carry a hydrogen atom, or a linkergroup as described below terminating in a hydrogen atom.

In a preferred embodiment of the invention, the receptor is bonded to afluorophore moiety to provide a glucose sensor molecule of formula (II):

wherein X, n, m and R₁ are as defined above.

Fl represents a fluorophore group. The fluorophore may be selected froma broad range of different functional groups. Examples of suitablefluorophore groups include those having π-electron systems, for examplenaphthyl, anthryl, pyrenyl, phenanthryl and perylenyl and derivativesthereof. Pyrenyl and anthryl, in particular pyrenyl groups and theirderivatives are preferred. Examples of suitable derivatives of thesefluorophores include those having one or more sulphonyl substituents,for example two or three sulphonyl substituents, in particular thosedescribed in WO 2010/116142, the contents of which are incorporatedherein by reference.

The fluorophore is linked to the receptor via a linking group, L₁.Typically L₁ consists of one or more alkylene groups, preferably C₁₋₆alkylene groups, and/or one or more arylene groups. The C₁₋₆ alkylenegroup is typically a straight chain group. It is typicallyunsubstituted. Preferred C₁₋₆ alkylene groups are straight chain C₁₋₄alkylene groups, e.g. methylene and ethylene, in particular methylene.The arylene group is typically a phenylene group. It is typicallyunsubstituted. In one embodiment, L₁ is a straight chain, unsubstitutedC₁₋₄ alkylene group, more preferably methylene or ethylene, mostpreferably methylene.

In the above formula (II), one nitrogen atom of the receptor is bondedto a linker L₂. L₂ typically consists of one or more alkylene groups,preferably C₁₋₆ alkylene groups, and/or one or more arylene groups. TheC₁₋₆ alkylene group is typically a straight chain group. It is typicallyunsubstituted. Preferred C₁₋₆ alkylene groups are straight chain C₁₋₄alkylene groups, e.g. methylene and ethylene, in particular methylene.The arylene group is typically a phenylene group. It is typicallyunsubstituted.

In one aspect, the group L₂ is selected from -alkylene-,-alkylene-arylene- and -alkylene-arylene-alkylene. For example, L₂ maybe a group of formula —(C₁₋₂ alkylene)-Ph-(C₀₋₂ alkylene)-, preferablymethylene-phenylene-methylene-.

R₄ typically represents a support material such as a hydrogel or adendrimer as described above. Alternatively, R₄ may be an end group suchas a hydrogen atom, where no support material is present.

In a further embodiment, R₄ represents an anchor group suitable forattaching the molecule to a support material. Thus, in this embodiment,the invention provides a precursor for the final supportmaterial-receptor complex described above. The anchor group is typicallya reactive group which is capable of forming a covalent bond with asecond reactive group on, for example, the support material to which itis to be attached. Suitable anchor groups include alkene, ester,aldehyde, amine and azide groups. These anchor groups may alternativelybe protected groups, e.g. protected aldehyde, amine or azide groups.Where an anchor group is present at position R₄, this may be used toreact with a support material (optionally after deprotection) to providea modified glucose sensor molecule wherein R₄ represents the supportmaterial.

Examples of preferred glucose sensor molecules of formula (II) are:

wherein Fl is a fluorophore as defined above, and R₄ is a supportmaterial, an anchor group suitable for attaching the molecule to asupport material, or a hydrogen atom. Preferably, Fl is pyrene or aderivative thereof and R₄ is a support material or an anchor groupselected from aldehyde, alkene, ester, azide or amine.

The glucose sensor molecules of the present invention can be preparedaccording to Scheme 1 below. Scheme 1 provides an exemplary synthesis inwhich X is O and R₄ is an anchor group.

In steps (a) and (b), the amine groups are protected with differentprotecting groups. Here Boc and Cbz are exemplified, but alternativeprotecting groups could be used, as long as the synthesis uses differentprotecting groups at each amine. A Williamson ether type reaction isthen carried out in step (c). Exemplary reaction conditions for steps(a) to (c) are as follows: a) 2-Ethanolamine, Boc₂O, DCM; b)3-bromopropylamine hydrobromide, benzyl chloroformate, 15% aqueous NaOH;c) ^(t)Bu₄NI, 20% aqueous NaOH, DCM.

The di-protected molecule (Va) then undergoes successive deprotectionand reductive amination in steps (d) to (h) to yield the final glucosesensor molecule (IIa). Exemplary reaction conditions are as follows: d)Pd/C, THF/MeOH sat. NH₃, H₂; e) activated -L₂-R₄ (e.g. compound 1, 4 or8 described below), MeOH, ii) NaBH₄; f) TFA, DCM, 0° C.; g) i) activatedFl-L₁- (e.g. Pyrene-1-carboxaldehyde), MeOH/THF, ii) NaBH₄; h) i)potassium 2-formylphenyltrifluoroborate, NaBH(OAc)₃, DIPEA, THF, ii)LiOH, MeCN/H₂O %.

Scheme 2 below depicts an alternative synthesis of the compounds of theinvention in which X is NH:

The skilled person would be able to adapt the above schemes to providethe corresponding compounds in which X is S, X is NR₂, wherein R₂ isother than H or wherein X is CHR₃. For sensors in which R₁ forms a ringtogether with R₂ or R₃, for example the compounds of formula (Ia),scheme 2 can be adapted by replacing

The activated Fl-L₁-compound is typically a compound in which theterminal carbon atom in an alkylene chain or arylene group of L₁ bears areactive group capable of reacting with an amine. Aldehyde is an exampleof such a reactive group. Activated Fl-L₁-compounds are commerciallyavailable or could be prepared by the skilled person using techniquesknown in the art.

Schemes 3 and 4 below provide an exemplary synthesis of activated -L₂-R₄compounds wherein L₂ is methylene-phenylene-methylene and R₄ is ananchor group. Compounds 1, 4 and 8 are each examples of suitableactivated -L₂-R₄ compounds in which the anchor group R₄ is a protectedaldehyde 1, an alkene 27, a protected amine 8 or azide 4.

Further detail regarding the synthesis of an exemplary glucose sensorcompound of the invention is provided in Example 1.

The above synthetic schemes provide glucose sensor compounds having ananchor group at R₄. The skilled person would be able to provide suitablealternative starting materials in order to provide corresponding glucosesensor compounds having a hydrogen atom at R₄. In order to provide asupport material at R₄, the compounds of formula (II) having an anchorgroup are reacted with an activated support material. The activatedsupport material has a reactive group capable of forming a bond with theanchor group (e.g. an amine group).

The present invention also provides a method of detecting or quantifyingthe amount of glucose in an analyte, the method comprising contactingthe analyte with a glucose receptor having a binding site of formula (I)as set out above. Typically, the analyte is contacted with a glucosesensor compound of formula (Ia) as set out above, in particular aglucose sensor compound of formula (Ia) in which R is a hydrogen atom ora support material. The glucose in the analyte binds to the binding sitein a selective manner. The binding of glucose causes a perturbation ofthe fluorescence of the fluorophore which can be detected. This can beachieved by detecting a change in the intensity of the fluorophore, orby detecting a change in the lifetime of the fluorophore.

The present invention is described below with reference to particularExamples. The invention is not intended to be limited to theseparticular Examples.

EXAMPLES Example 1 Synthesis of Glucose Sensor Molecule 1.11-(Diethoxymethyl)-4-(hydroxymethyl)benzene, 1

4-(Diethoxymethyl)benzaldehyde (10 g, 48 mmol) was dissolved in methanol(200 ml) and cooled to 0° C. NaBH₄ (4.54 g, 120 mmol, 2.5 eq) was thenadded slowly and the reaction mixture was stirred for 1 hour, afterwhich the solvent was evaporated. The residue obtained was dissolved inethyl acetate (100 ml) and water (100 ml), the phases were separated andthe organic phase was washed with water (100 ml), dried over magnesiumsulphate, and evaporated to yield 1 as a clear oil (10.09 g, 48 mmol,100%). ¹H NMR (300 MHz, CDCl₃) δ=7.48 (d, ³J(H,H)=8.1 Hz, 2H, ArCH α toCH₂OH), 7.37 (d, ³J(H,H)=8.1 Hz, 2H, ArCH α to CH(OEt)₂), 5.51 (s, 1H,CH(OEt)₂), 4.70 (d, ³J(H,H)=5.9 Hz, 2H, CH₂OH), 3.61 (dq, ³J(H,H)=7.1Hz, ²J(H,H)=9.5 Hz, 2H, CH₂CH₃), 3.56 (dq, ³J(H,H)=7.1 Hz, ²J(H,H)=9.5Hz, 2H, CH₂CH₃), 1.75 (t, ³J(H,H)=5.9 Hz, 1H, CH₂OH), 1.24 (t,³J(H,H)=7.1 Hz, 6H, CH₂CH₃); ¹³C NMR (75 MHz, CDCl₃) δ=140.9 (ArCCH₂OH),138.6 (ArCCH(OEt)₂), 126.9 (ArCH α to ArCCH₂OH), 126.8 (ArCH α toArCCH(OEt)₂), 101.3 (CH(OEt)₂), 65.1 (CH₂OH), 61.0 (OCH₂CH₃), 15.2(OCH₂CH₃).

1.2 4-(Hydroxymethyl)benzaldehyde, 2

Alcohol 1 (10.09 g, 48 mmol) was dissolved in a mixture of THF (100 ml)and 2 M HCl (100 ml) and stirred for 1 hour. The solvent was evaporatedand the residue obtained was dissolved in ethyl acetate (100 ml) andwater (100 ml). The phases were separated and the organic phase waswashed with water (100 ml), dried over magnesium sulphate, andevaporated to yield 2 as a white solid (6.54 g, 48 mmol, 100%).R_(f)=0.54 (ethyl acetate/chloroform, 1:1); v_(max)=3327, 1689, 1607,1206, 1010, 823 cm⁻¹; ¹H NMR (250 MHz, CDCl₃) δ=10.02 (s, 1H, CHO), 7.89(d, ³J(H,H)=8.1 Hz, 2H, ArCH α to CHO), 7.54 (d, ³J(H,H)=8.1 Hz, 2H,ArCH α to CH₂OH), 4.82 (d, ³J(H,H)=5.9 Hz, 2H, CH₂OH), 1.94 (t,³J(H,H)=5.9 Hz, 1H, CH₂OH); ¹³C NMR (75 MHz, CDCl₃) δ=192.0 (CHO), 147.7(ArCCOH), 135.7 (ArCCHO), 130.0 (ArCH α to ArCCHO), 127.0 (ArCH α toArCCH₂OH), 64.6 (CH₂OH); HRMS (ESI⁻): m/z calculated for C₈H₇O₂ [M−H]⁻:135.0446, found 135.0448; elemental analysis calcd (%) for C₈H₈O₂(136.15): C, 70.57; H, 5.92. found: C, 70.70; H 6.00.

1.3 4-(Bromomethyl)benzaldehyde, 3

Alcohol 2 (6.46 g, 47.5) was dissolved in DCM (100 ml) before HBr inacetic acid (33 wt %, 42 ml, 243, 5 eq) was added and stirred overnight.Water (100 ml) was added to the reaction mixture and the phases wereseparated and the organic phase obtained was washed with a NaOH solution(2 M, 2×100 ml), dried over Na₂SO₄, and evaporated. The residue waswashed through a silica plug to yield 3 as a white solid (9.04 g, 45.4mmol, 95%). R_(f)=0.77 (DCM); m.p.=100° C. (recrystallised from hexane);v_(max)=1682, 1604, 1209, 1200, 830, 770, 726 cm⁻¹; ¹H NMR (300 MHz,CDCl₃) δ=10.02 (s, 1H, CHO), 7.87 (d, ³J(H,H)=8.2 Hz, 2H, ArCH α toCHO), 7.56 (d, ³J(H,H)=8.2 Hz, 2H, ArCH α to CH₂Br), 4.52 (s, 21-1,CH₂Br); ¹³C NMR (75 MHz, CDCl₃) δ=191.5 (CHO), 144.2 (ArCCBr), 136.2(ArCCHO), 130.2 (ArCH α to ArCCHO), 129.7 (ArCH α to ArCCH₂Br), 31.9(CH₂Br); HRMS (ESI⁻): m/z calculated for C₈H₆BrO [M−H]⁻: 196.9602, found196.9602; elemental analysis calcd (%) for C₈H₇BrO (199.04): C, 48.27;H, 3.54. found: C, 47.40; H, 3.53.

1.4 4-(Azidomethyl)benzaldehyde, 4

Bromide 3 (180 mg, 0.90 mmol) was dissolved in DMF (10 ml). Sodium azide(88 mg, 1.35 mmol) was added. The reaction mixture was then heated at60° C. for an hour. The reaction mixture was allowed to cool and wasdissolve in ethyl acetate (150 ml) and H₂O (150 ml). The phases wereseparated and the organic phase was washed again with water (2×150 ml).The organic phase was dried over sodium sulphate, and evaporated underreduced pressure to yield 4 as an oil (134 mg, 0.83 mmol, 92%).R_(f)=0.70 (DCM); v_(max)=2094, 1694, 1607, 1207, 1167, 812, 773 cm⁻¹;¹H NMR (300 MHz, CDCl₃) δ=10.02 (s, 1H, CHO), 7.90 (d, ³J(H,H)=7.9 Hz,2H, ArCH α to CHO), 7.48 (d, ³J(H,H)=7.9 Hz, 2H, ArCH α to CH₂N₃), 4.45(s, 2H, CH₂N₃); ¹³C NMR (75 MHz, CDCl₃) δ=191.6 (CHO), 142.1 (ArCCH₂N₃),136.2 (ArCCHO), 130.2 (ArCH α to ArCCHO), 128.4 (ArCH α to ArCCH₂N₃),54.2 (CH₂N₃); HRMS (ESI⁺): m/z calculated for C₈H₇N₃ONa [M+Na]⁺:184.0481, found 184.0497.

1.5 1-(Azidomethyl)-4-(hydroxymethyl)benzene, 5

Azide 4 (2.0 g, 12.4 mmol) was dissolved in MeOH (50 ml) and cooled to0° C. before NaBH₄ was added slowly and the reaction mixture was stirredfor 1 hour, after which the solvent was evaporated. The residue obtainedwas dissolved in ethyl acetate (50 ml) and water (50 ml), the phaseswere separated and the organic phase was washed with water (100 ml),dried over Na₂SO₄, and evaporated to yield 5 as a clear oil (1.96 g,12.0 mmol, 97%). R_(f)=0.45 (DCM); v_(max)=cm⁻¹; ¹H NMR (300 MHz, CDCl₃)δ=7.40 (d, ³J(H,H) 8.2 Hz, 2H, ArCH α to CH₂OH), 7.33 (d, ³J(H,H)=8.2Hz, 2H, ArCH α to CH₂N₃), 4.71 (s, 2H, CH₂OH), 4.34 (s, 2H, CH₂N₃), 1.80(bs, 1H, OH); ¹³C NMR (75 MHz, CDCl₃) δ=141.0 (ArCCH₂OH), 134.7(ArCCH₂N₃), 128.5 (ArCH α to CH₂N₃), 127.4 (ArCH α to CH₂OH), 64.9(CH₂OH), 54.5 (CH₂N₃).

1.6 1-(Aminomethyl)-4-(hydroxymethyl)benzene, 6

Azide 5 (1.96 g, 12.0 mmol) and PPh₃ (6.50 g, 24.8 mmol, 2.05 eq) weredissolved in THF (25 ml) and heated at 60° C. for 1 hour. Water (4.5 ml,248 mmol, 20 eq) was added and the reaction was heated overnight. Thesolvent was evaporated and the residue obtained was purified by flashchromatography (eluent DCM to 4:1 DCM/methanol saturated with NH₃) toyield 6 as a white solid (1.44 g, 10.5 mmol, 85%). R_(f)=0.05 (9:1,DCM/MeOH sat. NH₃); v_(max)=cm⁻¹; ¹H NMR (300 MHz, CDCl₃) δ=7.35 (d,³J(H,H)=8.4 Hz, 2H, ArCH α to CH₂NH₂), 7.30 (d, ³J(H,H)=8.4 Hz, 2H, ArCHα to CH₂OH), 4.67 (s, 2H, CH₂OH), 3.85 (s, 2H, CH₂NH₂), 1.68 (bs, 3H,OH, NH₂); ¹³C NMR (75 MHz, CDCl₃) δ=142.6 (ArCCH₂NH₂), 139.6 (ArCCH₂OH),127.3 (ArCH), 127.2 (ArCH), 65.0 (CH₂OH), 46.2 (CH₂NH₂); HRMS (ESI⁺):m/z calculated for C₈H₁₂NO [M+H]⁺: 138.0913, found 138.0933.

1.7 1-^(t)Butoxycarbonylaminomethyl-4-hydroxymethyl benzene, 7

Amine 6 (1.44 g, 10.5 mmol) was dissolved in CHCl₃ (50 ml) and Boc₂O(2.29 g, 10.5 mmol, 1 eq) was added slowly. The reaction was stirredunder nitrogen overnight before the solvent was evaporated and theresidue obtained was dissolved in ethyl acetate (50 ml). This solutionwas washed with a citric acid solution (3×50 ml), brine (50 ml), driedover Na₂SO₄, and evaporated to yield 7 as a white solid (2.20 g, 9.2mmol, 88%). R_(f)=0.57 (DCM/MeOH sat. NH₃, 8:2); v_(max)=cm⁻¹; ¹H NMR(300 MHz, CDCl₃) δ=7.33 (d, ³J(H,H)=8.2 Hz, 2H, ArCH α to CH₂OH), 7.26(d, ³J(H,H)=8.2 Hz, 2H, ArCH α to CH₂NHBoc), 4.86 (bs, 1H, NHBoc), 4.68(s, 2H, CH₂OH), 4.30 (d, ³J(H,H)=5.7 Hz, 2H, CH₂NHBoc), 1.96 (bs, 1H,OH), 1.46 (s, 9H, C(CH₃)₃); ¹³C NMR (75 MHz, CDCl₃) δ=155.9 (CO), 140.0(ArCCH₂OH), 138.3 (ArCCH₂NHBoc), 127.6 (ArCH α to CH₂NHBoc), 127.2 (ArCHα to CH₂OH), 85.2 (C(CH₃)₃), 65.0 (CH₂OH), 44.4 (CH₂NHBoc), 28.4(C(CH₃)₃); HRMS (ESI⁺): m/z calculated for C₁₃H₁₉NO₃Na [M+Na]⁺:260.1257, found 260.1253.

1.8 4-^(t)Butoxycarbonylaminomethyl-benzaldehyde, 8

Alcohol 7 (2.20 g, 9.2 mmol) was dissolved in DCM (100 ml) and MnO₂(8.18 g, 92 mmol, 10 eq) was added and the resulting suspension wasstirred for 3 hours. The reaction mixture was then filtered throughcelite and evaporated to yield Error! Reference source not found. as awhite solid (2.18 g, 9.2 mmol, 100%). R_(f)=0.62 (DCM/Ethyl acetate,9:1); v_(max)=cm⁻; ¹H NMR (300 MHz, CDCl₃) δ=10.0 (CHO), 7.85 (d,³J(H,H)=8.1 Hz, 2H, ArCH α to CHO), 7.45 (d, ³J(H,H)=8.1 Hz, 2H, ArCH αto CH₂NHBoc), 4.99 (bs, 1H, NHBoc), 4.40 (d, ³J(H,H)=5.7 Hz, 2H,CH₂NHBoc), 1.47 (s, 9H, C(CH₃)₃); ¹³C NMR (75 MHz, CDCl₃) δ=191.9 (CHO),155.9 (CO), 145.9 (ArCCH₂NHBoc), 135.5 (ArCCHO), 130.1 (ArCH α to CHO),127.6 (ArCH α to CH₂NHBoc), 85.1 (C(CH₃)₃), 44.3 (CH₂NHBoc), 28.3(C(CH₃)₃); HRMS (ESI⁺): m/z calculated for C₁₃H₁₉NO₃Na [M Na]⁺:258.1101, found 258.1094. NMR data consistent with published data.⁶

1.9 Potassium 2-Formylphenyltrifluoroborate, 10

2-Formyl boronic acid (2.0 g, 13.3 mmol) was dissolved in MeOH (5 ml)before KHF₂ (4.16 g, 53.3 mmol) was added. A white precipitate formedand the solvent was evaporated 30 minutes after the addition of theKHF₂. The residue obtained was extracted with MeOH/acetone (1:4, 4×25ml) and evaporated before being recrystalised from diethyl ether toyield 10 as a white crystalline solid (2.82 g, 13.3 mmol, 100%). ¹H NMR(300 MHz, DMSO-d₆) δ=10.44 (s, 1H, CHO), 7.67 (d, ³J(H,H)=7.4 Hz, 1H,ArCH), 7.61 (d, ³J(H,H)=7.4 Hz, 1H, ArCH), 7.39 (t, ³J(H,H)=7.4 Hz, 1H,ArCH (3), 7.23 (t, ³J(H,H)=7.4 Hz, 1H, ArCH); ¹¹B NMR (96 MHz, DMSO-d₆)δ=8.5; ¹³C NMR (75 MHz, DMSO-d₆) δ=197.0 (CHO), 132.8 (ArCH), 132.7(ArCCHO), 131.9 (ArCH), 125.8 (ArCH), 124.4 (ArCH); HRMS (ESI⁺): m/zcalculated for C₇H₅OBF₃K [M−K]⁻: 173.0386, found 173.0396.

1.10 2-(^(t)Butoxycarbonylamino)ethanol, 11

A solution of Boc₂O (33.6 g, 154 mmol, 1.1 eq) in dry DCM (40 ml) wasadded dropwise to a solution of 2-aminoethanol (8.55 g, 140 mmol) in dryDCM (180 ml) at r.t. under nitrogen. The reaction mixture was stirredovernight then washed with a saturated NaHCO₃ solution (3×200 ml). Theorganic layer was dried over Na₂SO₄ and evaporated. The oil obtained wasdistilled under reduced pressure to give 11 as a colourless clear oil(16.0 g, 99 mmol, 71%). ¹H NMR (300 MHz, CDCl₃) δ=5.06 (bs, 1H, NHBoc),3.67 (t, ³J(H,H)=5.1 Hz, 2H, CH₂OH), 3.26 (t, ³J(H,H)=5.1 Hz, 2H,CH₂NHBoc), 1.43 (s, 9H, C(CH₃)₃), 1.25 (s, 1H, CH₂OH); ¹³C NMR (75 MHz,CDCl₃) δ=156.8 (CO), 79.6 (C(CH₃)₃), 62.3 (CH₂OH), 43.2 (CH₂NHBoc), 28.3(C(CH₃)₃); HRMS (ESI⁺): m/z calculated for C₇H₁₆NO₃ [M+H]⁺: 162.1130,found 162.1123, m/z calculated for C₇H₁₅NO₃Na [M+Na]⁺: 184.0950, found184.0942.

1.11 3-(Benzyloxycarbonylamino)propyl bromide, 12

3-Bromopropylamine hydrobromide (5.0 g, 22.8 mmol) was dissolved in anaqueous NaOH solution (15 wt %, 80 ml) and cooled to 0° C. undernitrogen before benzyl chloroformate was added dropwise. The reactionwas left to stir overnight then ethyl acetate (100 ml) was added and thephases were separated. The organic phase was further washed with a HClsolution (2 M, 100 ml), a NaOH solution (2 M, 100 ml), brine (100 ml),dried over Na₂SO₄, and evaporated to yield 12 as a clear oil (6.22 g,22.8 mmol, 100%). R_(f)=0.31 (8:2 Hexane/Ethyl acetate); ¹H NMR (300MHz, CDCl₃) δ=7.40-7.33 (m, 5H, ArCH), 5.11 (s, 2H, CH₂Ph), 4.94 (bs,1H, NHCbz), 3.45 (t, ³J(H,H)=6.4 Hz, 2H, CH₂Br), 3.36 (q, ³J(H,H)=6.4Hz, 2H, CH₂NHCbz), 2.08 (q, ³J(H,H)=6.4 Hz, 2H, CH₂CH₂CH₂); ¹³C NMR (75MHz, CDCl₃) δ=156.4 (CO), 136.4 (ArCCH₂), 128.5 (ArCH), 128.2 (ArCH),127.0 (ArCH), 66.8 (CH₂Ph), 39.4 (CH₂NHCbz), 32.4 (CH₂CH₂CH₂), 30.6(CH₂Br); HRMS (ESI⁺): m/z calculated for C₁₁H₁₄BrNO₂Na [M+Na]⁺:294.0106, found 294.0099.

1.12 2-(^(t)Butoxycarbonylamino)ethyl 3-(benzyloxycarbonylamino)propylether, 13

Alcohol 11 (1.0 g, 6.20 mmol), bromide 12 (2.19 g, 8.06 mmol, 1.3 eq),and ^(t)BuNI (2.98 g, 8.06 mmol, 1.3 eq), were dissolved in DCM (50 ml)and an aqueous solution of NaOH (20 wt %, 50 ml) was added and thereaction was stirred overnight under nitrogen. The phases were separatedand the aqueous phase was washed with DCM (2×50 ml), the organic phaseswere combined, dried over Na₂SO₄ and evaporated. The residue obtainedwas purified by flash chromatography (eluent DCM to ethyl acetate) toyield Error! Reference source not found. as a clear oil (568 mg, 1.61mmol, 26%). R_(f)=0.34 (9:1 DCM/MeOH saturated with NH₃); ¹H NMR (300MHz, CDCl₃) δ=7.38-7.30 (m, 5H, ArCH), 5.13 (bs, 1H, NHX), 5.10 (s, 2H,CH₂Ph), 4.97 (bs, 1H, NHX), 3.48 (m, 4H, CH₂O), 3.30 (m, 4H, CH₂N), 1.77(q, ³J(H,H)=6.1 Hz, 2H, CH₂CH₂CH₂), 1.44 (s, 9H, C(CH₃)₃); ¹³C NMR (75MHz, CDCl₃) δ=156.4 (CO), 156.0 (CO), 136.6 (ArC), 128.5 (ArCH), 128.1(ArCH), 128.0 (ArCH), 79.3 (C(CH₃)₃), 70.0 (BocHNCH₂CH₂O), 68.7(OCH₂CH₂CH₂), 66.5 (CH₂Ph), 40.3 (CH₂NHBoc), 38.7 (CH₂CH₂CH₂NHCbz), 29.7(CH₂CH₂CH₂), 28.4 (C(CH₃)₃); HRMS (ESI⁺): m/z calculated for C₁₈H₂₉N₂O₅[M+H]⁺: 353.2071, found 353.2072, m/z calculated for C₁₈H₂₈N₂O₅Na[M+Na]: 375.1890, found 375.1890.

1.13 2-(^(t)Butoxycarbonylamino)ethyl 3-aminopropyl ether, 14

Pd on C (200 mg) was activated by heating at 300° C. under vacuum for 4hours before a solution of ether 13 (568 g, 1.61 mmol) in THF and MeOHsaturated with NH3 (1:1, 100 ml) was added. The flask was evacuated andfilled with hydrogen (1 atm) and the reaction was stirred for 1 hour.The reaction mixture was then filtered over celite and washed with ethylacetate before the filtrate evaporated. The residue obtained waspurified by flash chromatography (eluent DCM to DCM/methanol saturatedwith NH₃, 19:1) to yield Error! Reference source not found. as a clearoil (334 mg, 1.57 mmol, 98%). R_(f)=0.65 (17:3 DCM/MeOH saturated withNH₃); ¹H NMR (300 MHz, CDCl₃) δ=5.03 (bs, 1H, NHBoc), 3.48 (t,³J(H,H)=5.3 Hz, 2H, OCH₂CH₂CH₂NH₂), 3.43 (t, ³J(H,H)=6.2 Hz, 2H,OCH₂CH₂NHBoc), 3.25 (q, ³J(H,H)=5.3 Hz, 2H, OCH₂CH₂NHBoc), 2.75 (t,³J(H,H)=6.8 Hz, 2H, OCH₂CH₂CH₂NH₂), 1.67 (quintet, ³J(H,H)=6.5 Hz, 2H,OCH₂CH₂CH₂CH₂NH₂), 1.40 (s, 9H, C(CH₃)₃), 1.27 (bs, 2H, NH₂); ¹³C NMR(75 MHz, CDCl₃) δ=155.9 (CO), 79.0 (C(CH₃)₃), 69.7 (OCH₂CH₂NHBoc), 69.0(OCH₂CH₂CH₂NH₂), 40.3 (OCH₂CH₂NHBoc), 39.4 (OCH₂CH₂CH₂NH₂), 33.2(OCH₂CH₂CH₂NH₂), 28.3 (C(CH₃)₃); HRMS (ESI⁺): m/z calculated forC₁₀H₂₃N₂O₃ [M+H]⁺: 219.1703, found 219.1690.

1.14 2-(^(t)Butoxycarbonylamino)ethyl3-N-(4-(azidomethyl)benzyl)-aminopropyl ether, 15

Amine 14 (270 mg, 1.24 mmol) and aldehyde 4 (219 mg, 1.36 mmol, 1.1 eq)were dissolved in methanol (50 ml) and stirred overnight. The reactionmixture was then cooled to 0° C. and NaBH₄ (117 mg, 3.1 mmol, 2.5 eq)was then added slowly and the reaction mixture was stirred for 2 hours,after which the solvent was evaporated. The residue obtained wasdissolved in DCM (50 ml) and water (50 ml), the phases were separatedand the aqueous phase was extracted with DCM (2×50 ml), dried overNa₂SO₄, and evaporated. The crude product was purified by flashchromatography (eluent DCM to DCM/methanol saturated with NH₃, 97:3)yielding 15 as a clear oil (360 mg, 0.99 mmol, 80%). R_(f)=0.63 (97:3DCM/MeOH saturated with NH₃); ¹H NMR (300 MHz, CDCl₃) δ=7.34 (d,³J(H,H)—-8.2 Hz, 2H, ArCH α to CH₂NH), 7.27 (d, ³J(H,H)=8.2 Hz, 2H, ArCHα to CH₂N₃), 5.03 (bs, 1H, NHBoc), 4.32 (s, 2H, CH₂N₃), 3.80 (s, 2H,CH₂NH), 3.52 (t, ³J(H,H)=6.2 Hz, 2H, OCH₂CH₂CH₂NH), 3.47 (t, ³J(H,H)=5.2Hz, 2H, OCH₂CH₂NHBoc), 3.29 (q, ³J(H,H)=5.3 Hz, 2H, OCH₂CH₂NHBoc), 2.71(t, ³J(H,H)=6.8 Hz, 2H, OCH₂CH₂CH₂NH), 1.78 (quintet, ³J(H,H)=6.5 Hz,2H, OCH₂CH₂CH₂NH), 1.59 (bs, 1H, CH₂NHCH₂), 1.44 (s, 9H, C(CH₃)₃); ¹³CNMR (75 MHz, CDCl₃) δ=155.9 (CO), 140.6 (ArCCH₂NH), 133.9 (ArCCH₂N₃),128.5 (ArCH α to CH₂NH), 128.3 (ArCH α to CH₂N₃), 79.2 (C(CH₃)₃), 69.7(OCH₂CH₂NHBoc), 69.4 (OCH₂CH₂CH₂NH), 54.5 (CH₂N₃), 53.6 (ArCH₂NH), 46.6(OCH₂CH₂CH₂NH), 40.4 (OCH₂CH₂NHBoc), 29.9 (OCH₂CH₂CH₂NH), 28.4(C(CH₃)₃); HRMS (ESI⁺): m/z calculated for C₁₈H₃₀N₅O₃ [M+H]⁺: 364.2343,found 364.2354.

1.15 2-Aminoethyl 3-N-(4-(azidomethyl)benzyl)-aminopropyl ether, 16

Boc protected amine 15 (360 mg, 0.99 mmol) was dissolved in dry DCM (10ml) under nitrogen and cooled to 0° C. Trifluoroacetic acid (2 ml) wasadded and the reaction mixture was stirred for an hour, after which thesolvent was evaporated. The residue obtained was dissolved in DCM (30ml) and NaOH (2 M, 50 ml), the phases were separated and the aqueousphase was extracted with DCM (2×30 ml), dried over Na₂SO₄, andevaporated yielding 16 as a clear oil (219 mg, 0.83 mmol, 84%).R_(f)=0.40 (9:1 DCM/MeOH saturated with NH₃); ¹H NMR (300 MHz, CDCl₃)δ=7.34 (d, ³J(H,H)=8.2 Hz, 2H, ArCH α to CH₂NH), 7.27 (d, ³J(H,H)=8.2Hz, 2H, ArCH α to CH₂N₃), 4.32 (s, 2H, CH₂N₃), 3.80 (s, 2H, CH₂NH), 3.53(t, ³J(H,H)=6.2 Hz, 2H, OCH₂CH₂CH₂NH), 3.44 (t, ³J(H,H)=5.2 Hz, 2H,OCH₂CH₂NH₂), 2.83 (t, ³J(H,H)=5.2 Hz, 2H, OCH₂CH₂NH₂), 2.73 (t,³J(H,H)=6.9 Hz, 2H, OCH₂CH₂CH₂NH), 1.80 (quintet, ³J(H,H)=6.5 Hz, 2H,OCH₂CH₂CH₂NH), 1.46 (bs, 3H, NH); ¹³C NMR (75 MHz, CDCl₃) δ=140.7(ArCCH₂NH), 133.9 (ArCCH₂N₃), 128.5 (ArCH α to CH₂NH), 128.3 (ArCH α toCH₂N₃), 73.0 (OCH₂CH₂NH₂), 69.5 (OCH₂CH₂CH₂NH), 54.5 (CH₂N₃), 53.6(ArCH₂NH), 46.8 (OCH₂CH₂CH₂NH), 41.9 (OCH₂CH₂NH₂), 30.0 (OCH₂CH₂CH₂NH);HRMS (ESI⁺): m/z calculated for C₁₃H₂₂N₅O [M+H]⁺: 264.1819, found264.1797.

1.16 2-(pyren-1-ylmethylamino)ethyl3-N-(4-(azidomethyl)benzyl)-aminopropyl ether, 17

Amine 16 (220 mg, 0.83 mmol) and 1-pyrene carboxaldehyde (230 mg, 1.0mmol, 1.2 eq) were dissolved in methanol and THF (1:1, 60 ml) and werestirred over night. The reaction mixture was cooled to 0° C. and NaBH₄(157 mg, 4.15 mmol, 5 eq) was added and the solvents was evaporatedafter stirring for a further hour. The residue obtained was dissolved inDCM (30 ml) and H₂O (30 ml), the phases were separated and the aqueousphase was extracted with DCM (2×30 ml), dried over Na₂SO₄, andevaporated. The crude product was purified by flash chromatography(eluent DCM to DCM/methanol saturated with NH₃, 98:2) yielding 17 as ayellow oil (228 mg, 0.48 mmol, 58%). R_(f)=0.80 (9:1 DCM/MeOH saturatedwith NH₃); ¹H NMR (300 MHz, CDCl₃) δ=8.40 (d, ³J(H,H)=9.3 Hz, 1H,Pyrene-ArCH), 8.22 (m, 1H, Pyrene-ArCH), 8.20 (m, 1H, Pyrene-ArCH), 8.17(d, ³J(H,H)=7.8 Hz, 1H, Pyrene-ArCH), 8.15 (d, ³J(H,H)=9.3 Hz, 1H,Pyrene-ArCH), 8.07 (s, 2H, Pyrene-ArCH), 8.05 (d, ³J(H,H)=7.6 Hz, 1H,Pyrene-ArCH), 8.03 (d, ³J(H,H)=10.6 Hz, 1H, Pyrene-ArCH), 7.29 (d,³J(H,H)=8.1 Hz, 2H, ArCH α to CH₂NH), 7.21 (d, ³J(H,H)=8.1 Hz, 2H, ArCHα to CH₂N₃), 4.52 (s, 2H, CH₂Pyrene), 4.26 (s, 2H, CH₂N₃), 3.73 (s, 2H,CH₂NH), 3.64 (t, ³J(H,H)=5.2 Hz, 2H, OCH₂CH₂NH), 3.55 (t, ³J(H,H)=6.2Hz, 2H, OCH₂CH₂CH₂NH), 3.00 (t, ³J(H,H)=5.2 Hz, 2H, OCH₂CH₂NH₂), 2.71(t, ³J(H,H)=6.9 Hz, 2H, OCH₂CH₂CH₂NH), 1.82 (quintet, ³J(H,H)=6.5 Hz,2H, OCH₂CH₂CH₂NH), 1.79 (bs, 2H, NH); ¹³C NMR (75 MHz, CDCl₃) δ=140.4(ArCCH₂NH), 133.7 (ArCCH₂N₃), 133.6 (Pyrene-ArC), 131.0 (Pyrene-ArC),130.6 (Pyrene-ArC), 130.4 (Pyrene-ArC), 128.8 (Pyrene-ArC), 128.1 (ArCHα to CH₂NH), 128.0 (ArCH α to CH₂N₃), 127.3 (Pyrene-ArCH), 127.2(Pyrene-ArCH), 126.8 (Pyrene-ArCH), 126.7 (Pyrene-ArCH), 125.6(Pyrene-ArCH), 124.8 (Pyrene-ArCH), 124.8 (Pyrene-ArC), 124.7(Pyrene-ArCH), 124.6 (Pyrene-ArC), 124.4 (Pyrene-ArCH), 123.0(Pyrene-ArCH), 70.0 (OCH₂CH₂NH), 69.3 (OCH₂CH₂CH₂NH), 54.2 (CH₂N₃), 53.3(ArCH₂NH), 51.4 (CH₂Pyrene), 49.0 (OCH₂CH₂NH), 46.5 (OCH₂CH₂CH₂NH), 29.8(OCH₂CH₂CH₂NH); HRMS (ESI⁺): m/z calculated for C₃₀H₃₂N₅O [M+H]⁺:478.2601, found 478.2570.

1.17 (N-(pyren-1-ylmethyl)-N-(benzyl-2-boronic acid)-aminoethyl)(N′-(4-(azidomethyl)benzyl)-N′-(benzyl-2′-boronicacid)-3-aminopropyl)ether, 18

Diamine 17 (228 mg, 0.48 mmol), potassium 2-formylphenyltrifluoroborate(213 mg, 1.00 mmol, 2.1 eq), and sodium triacetoxyborohydride (224 mg,1.06 mmol, 2.2 eq) were dissolved in dry THF (20 ml) and DIPEA (836 ml,4.80 mmol, 10 eq) under a nitrogen environment and stirred for 3 days.NaBH₄ (36 mg, 0.96 mmol, 2 eq) was added before the solvent wasevaporated. The residue obtained was extracted with hot acetone andevaporated before being suspended in acetonitrile/water (10:9) (50 ml).Lithium hydroxide (69 mg, 2.88 mmol, 6 eq) was added and the reactionwas stirred at room temperature overnight. The reaction mixture wasextracted with ethyl acetate (3×50 ml), the organic phases werecombined, dried over magnesium sulphate, and evaporated to yield 18 as ayellow solid (340 mg, 0.46 mmol, 96%). ¹H NMR (300 MHz, CDCl₃/CD₃OD90:10) δ=8.24-7.98 (m, 10H, ArCH), 7.89 (m, ArCH), 7.47-7.31 (m, 6H,ArCH), 7.24 (d, ³J(H,H)=8.3 Hz, 2H, ArCH α to CH₂N), 7.17 (d,³J(H,H)=8.3 Hz, 2H, ArCH α to CH₂N₃), 4.33 (s, 2H, CH₂Pyrene), 4.22 (s,2H, CH₂N₃), 3.98 (s, 2H, (C₆H₄BOH₂CH₂N), 3.69 (s, 2H, (C₆H₄BOH₂)CH₂N),3.54 (s, 2H, CH₂NH), 3.45 (t, ³J(H,H)=5.6 Hz, 2H, OCH₂CH₂N), 3.23 (t,³J(H,H)=6.1 Hz, 2H, OCH₂CH₂CH₂N), 2.79 (t, ³J(H,H)=5.6 Hz, 2H,OCH₂CH₂N), 2.51 (m, 2H, OCH₂CH₂CH₂NH), 1.76 (m, 2H, OCH₂CH₂CH₂NH); ¹¹BNMR (96 MHz, CDCl₃/CD₃OD 95:5) δ=−9.4 ¹³C NMR (75 MHz, CDCl₃/CD₃OD90:10) δ=143.8 (ArCH), 141.5 (ArC), 141.1 (ArC), 136.4 (ArCCH₂N), 135.8(ArCH), 135.7 (ArCH), 134.3 (ArCCH₂N₃), 131.1 (ArCH), 131.1 (ArC), 130.7(ArC), 130.6 (ArCH), 130.5 (ArC), 130.3 (ArC), 129.8 (ArCH), 129.7(ArC), 129.7 (ArCH α to CH₂N), 128.6 (ArCH), 128.0 (ArCH α to CH₂N₃),127.3 (ArCH), 127.3 (ArCH), 127.3 (ArCH), 127.2 (ArCH), 127.1 (ArCH),125.8 (ArCH), 125.1 (ArCH), 124.9 (ArCH), 124.7 (ArC), 124.5 (ArC),124.4 (ArCH), 123.1 (ArCH), 68.7 (OCH₂CH₂CH₂N), 67.9 (OCH₂CH₂N), 62.4((C₆H₄BOH₂)CH₂N), 61.0 ((C₆H₄BOH₂)CH₂N), 56.6 (ArCH₂N), 54.8(CH₂Pyrene), 54.1 (CH₂N₃), 53.3 (ArCH₂N), 52.0 (OCH₂CH₂N), 49.2(OCH₂CH₂CH₂N), 25.0 (OCH₂CH₂CH₂N); HRMS (ESI⁺): m/z calculated forC₄₄H₄₄B₂N₅O₄ [M+H−H₂O]⁺: 728.3574, found 728.3525.

Example 2 Binding Studies

Fluorescence titration studies were carried out with glucose sensormolecule 18 synthesised as described in Example 1 above, as well as withtwo comparative compounds 19 and 20:

Compound 19 was synthesised according to the procedure of Arimori, S.;Bell, M. L.; Oh, C. S.; Frimat, K. A.; James, T. D. Chem. Commun. 2001,1836. Compound 20 was synthesised in a similar manner, with appropriatemodification at position R₄.

For each sensor compound, titrations were carried out using D-glucose,D-fructose, D-mannose and D-galactose, according to the followingstandard procedure:

Stock solutions of carbohydrates were made up in an aqueous methanolicbuffer [52.1 wt % methanol, KCl (10.0 mM), KH₂PO₄ (2.75 mM) and Na₂HPO₄(2.75 mM)] at a pH of 8.21 and allowed to equilibrate overnight prior touse. Additions to receptor were performed using a procedure which kept[host] (i.e. [receptor]) and the total volume constant while raising[guest] (i.e. [carbohydrate]). Thus, receptor was added to a stockcarbohydrate solution to give [host]=0.1 or 1.0 μM. This solution wasused as titrant. A solution of guest in an aqueous methanolic buffer[52.1 wt % methanol, KCl (10.0 mM), KH₂PO₄ (2.75 mM) and Na₂HPO₄ (2.75mM)] at a pH of 8.21, also 0.1 or 1.0 μM, was placed in a fluorescencecell. For each addition, an aliquot of a certain volume was removed fromthe cell, and the same volume of titrant was then added.

The mixture was shaken then sonicated, and the fluorescence spectrumrecorded on a PerkinElmer LS 50B fluorescence spectrometer at roomtemperature. The excitation wavelength was set at 342 nm. Emissionchanges in counts per second (DCPS) were analysed according to a 1:1binding model, using a non-linear least squares curve-fitting programimplemented within Excel for calculation of a association constantK_(obs). In the case of D-fructose, an attempt to analyse the dataaccording to the 1:1 binding model yielded a very poor fit. Instead, theintensity changes were analysed according to a 1:1+1:2 binding model,using the program WinEqNMR.

Results:

TABLE 1 Observed 1:1 stability constants (K_(obs)), determinationcoefficient (r²), and fluorescence enhancement for receptors 18-20 (0.1μM) with D-glucose, D-fructose, D-galactose, and D-mannose. 19 20 18K_(obs)/M⁻¹ I_(∞)/I₀ K_(obs)/M⁻¹ I_(∞)/I₀ K_(obs)/M⁻¹ I_(∞)/I₀ D-Glucose962 ± 70 2.8 1072 ± 185 4.6 1476 ± 51  4.4 D-Galactose 657 ± 39 3.1 536± 42 1.8 243 ± 16 2.8 D-Fructose 784 ± 44 3.2 3542 ± 500 1.0 K_(obs1) =765 ± 260;  1.5 K_(obs2) = 210 ± 1553 D-Mannose 74 ± 3 2.8 101 ± 8  2.048 ± 4 2.5

The results are also depicted graphically in FIGS. 1 to 3. FIG. 1 showsthe relative fluorescence intensity versus carbohydrate concentrationprofile of 18 (0.1 μM, λ_(ex)=342 nm, λ_(em)≈377 nm) displayingphotoinduced electron transfer (PET) with (▪) D-glucose, () D-fructose,(♦) D-galactose, (▴) D-mannose. FIG. 2 provides corresponding resultsfor compound 20. FIG. 3 shows the relative fluorescence intensity versuscarbohydrate concentration profile of 19 from literature data.

The results show that the glucose sensor molecule of the invention hasgreater affinity and selectivity for D-glucose than compounds 19 and 20,which do not contain an oxygen atom in the carbon chain in the glucosebinding site.

Example 3 Attachment of Glucose Sensor Molecule to Hydrogel

A solution of AIPD (8 mg) in H₂O (700 μl) was added to a solution ofcompound 21 (16 mg), dimethylacrylamide (740 mg) and PEG-DMA 600 (1.40ml) in a aqueous TFA solution (5 mM, 700 μl) under nitrogen. Thissolution was then heated for 1 hour at 45° C. followed by 1.5 hours at60° C. to yield a boronic acid receptor containing hydrogel. Thepolymerisation was then quenched by submerging the reaction vessel inice water.

1. A glucose sensor comprising a glucose receptor having a binding siteof formula (I):

wherein X represents O, S, NR₂ or CHR₃; n is from 1 to 4; m is from 1 to4, and n+m is 5; R₂ represents hydrogen or C₁₋₄ alkyl; each R₁ is thesame or different and represents hydrogen, C₁₋₄ alkyl or C₃₋₇cycloalkyl; or R₁, together with an adjacent R₁, R₂ or R₃ group and thecarbon or nitrogen atoms to which they are attached, form a C₃₋₇cycloalkyl or a 5- or 6-membered heterocyclyl group, wherein when Xrepresents CHR₃, R₃ together with an adjacent R₁ group and the carbonatoms to which they are attached form a C₃₋₇ cycloalkyl group.
 2. Aglucose sensor according to claim 1, wherein X represents O, S or NR₂.3. A glucose sensor according to claim 2, wherein X represents O.
 4. Aglucose sensor according to claim 2, wherein each R₁ is the same ordifferent and represents hydrogen, C₁₋₄ alkyl or C₃₋₇ cycloalkyl; and R₂represents hydrogen or C₁₋₄ alkyl.
 5. A glucose sensor according toclaim 1, wherein at least four R₁ groups represent hydrogen.
 6. Aglucose sensor according to claim 1, wherein X represents O; n is from 1to 4; m is from 1 to 4 and n+m is 5; and each R₁ represents hydrogen. 7.A glucose sensor according to claim 6, wherein n is 2 or 3, m is 2 or 3and n+m is
 5. 8. A glucose sensor according to claim 1, wherein thebinding site is of formula (Ia):

wherein p is from 1 to 4; q is from 0 to 3, and p+q is 4; X is N or CH;each R₁ is the same or different and represents hydrogen, C₁₋₄ alkyl orC₃₋₇ cycloalkyl; and ring A is a C₃₋₇ cycloalkyl group or a 5- to7-membered heterocyclyl group.
 9. A glucose sensor according to claim 1,wherein the glucose receptor is bonded to a support material at one ofthe nitrogen atoms marked as N*:


10. A glucose sensor according to claim 9, wherein the support materialis a dendrimer or water-soluble polymer and the resulting complex ofreceptor and support material is in aqueous solution.
 11. A glucosesensor according to claim 1, wherein the sensor further comprises afluorophore, the fluorophore being associated with the glucose receptorsuch that when glucose is bound to the receptor, the fluorescence of thefluorophore is perturbed.
 12. A glucose sensor according to claim 1,wherein the sensor comprises a glucose sensor molecule of formula (II):

wherein X, n, m and R₁ are as defined in any one of one of claims 1 to8; Fl is a fluorophore; L₁ and L₂ are the same or different andrepresent a linker; and R₄ is a support material or a hydrogen atom. 13.A glucose sensor according to claim 12, wherein L₁ and L₂ are the sameor different and each consists of one or more C₁₋₆ alkylene groupsand/or one or more arylene groups.
 14. A glucose sensor according toclaim 13, wherein L₂ is a group of formula —(CH₂)_(s)-Ph-(CH₂)_(t)—,wherein s is 1 or 2, t is 0, 1 or 2 and Ph is phenylene; and/or L₁ ismethylene or ethylene.
 15. A glucose sensor according to claim 12,wherein the sensor comprises a glucose sensor molecule of formula (II′)or (II″):

wherein Fl is a fluorophore; and R₄ is a support material or a hydrogenatom.
 16. A glucose sensor according to any one of claim 12, wherein R₄is a support material and the support material is a polymeric matrix, awater-soluble polymer or a water soluble molecule selected fromdendrimers, cyclodextrins, cryptans and crown ethers.
 17. A glucosesensor molecule of formula (II):

wherein X, n, m and R₁ are as defined in any one of one claims 1 to 8;Fl is a fluorophore; L₁ and L₂ are the same or different and represent alinker as defined in any one of claims 12 to 14; and R₄ is a supportmaterial as defined in claim 12 or 16, a hydrogen atom or an anchorgroup suitable for attaching the sensor molecule to a support material.18. A glucose sensor molecule according to claim 17, wherein R₄ is ananchor group selected from an alkene, ester, aldehyde, amine or azidegroup.
 19. A process for the preparation of a glucose sensor molecule asclaimed in claim 17, which process comprises reductive amination of(III) in the presence of (IV), followed by deprotection of the boronicacid group:Fl-L₁-NH—(CHR₁)_(n)—X—(CHR₁)_(m)—NH-L₂-R₄  (III) wherein X, n, m and R₁are as defined in any one of one claims 1 to 8; Fl is a fluorophore; L₁and L₂ are the same or different and represent a linker as defined inany one of claims 12 to 14; and R₄ is a hydrogen atom or an anchor groupsuitable for attaching the sensor molecule to a support material;wherein B(PG) is a boronic acid group protected by a protecting group.20. A method of detecting or quantifying the amount of glucose in ananalyte, the method comprising contacting the analyte with a glucosereceptor comprising a binding site of formula (I):

wherein X, n, m and R₁ are as defined in any one of one claims 1 to 8.21. A method according to claim 20, which method is a fluorescencesensing method comprising detecting a change in the fluorescence of thesensor when glucose is bound to the glucose receptor.
 22. A methodaccording to claim 21, wherein the change in fluorescence is a change influorescence lifetime.
 23. A process according to claim 19, wherein thechange in fluorescence is a change in fluorescence intensity.
 24. Aglucose sensor according to claim 8, wherein each R1 representshydrogen.
 25. A process for the preparation of a glucose sensor moleculeas claimed in claim 17, which process comprises reductive amination of(III) in the presence of (IV), followed by deprotection of the boronicacid group and deprotection of R₄:Fl-L₁-NH—(CHR₁)_(n)—X—(CHR₁)_(m)—NH-L₂-R₄  (III) wherein X, n, m and R₁are as defined in any one of one claims 1 to 8; Fl is a fluorophore; L₁and L₂ are the same or different and represent a linker as defined inany one of claims 12 to 14; and R₄ is a hydrogen atom or an anchor groupsuitable for attaching the sensor molecule to a support material,wherein R₄ is protected by a protecting group;

wherein B(PG) is a boronic acid group protected by a protecting group.